Angiogenesis is an important biological process not only under physiological conditions, but also in association with diseases such as tumor angiogenesis, diabetic retinopathy, and chronic rheumatoid arthritis. In angiogenesis, many signaling systems such as vascular endothelial growth factor (VEGF) and its receptor, angiopoietin-Tie receptor, ephrin-Eph4 receptor are involved. Among them, VEGF and its receptor, inter alia, plays an important role by being involved in the proliferation of vascular endothelial cells and the acceleration of vascular permeability.
VEGF is a proliferation factor specific for endothelial cells which is a 34 to 45 kDa dimeric glycoprotein formed by subunits with a molecular weight of approximately 20 kDa. VEGF has a wide range of activities such as promotion of vasculogenesis, enhancement of vascular permeability, and other activities. VEGF belongs to a platelet-derived growth factor (PDGF) family which is a growth factor and it has a homology of approximately 18% at an amino acid level with chain A and chain B of PDGF. Moreover, VEGF comprises the eight conservative cysteine residues common to all growth factors belonging to the PDGF family.
VEGF exerts an influence over vascular endothelial cells by binding to specific high-affinity cell surface receptors. In endothelial cells, 150 and 130 kDA receptors are identified. VEGF receptors belong to a super family of receptor tyrosine kinase (RTKs) which is characterized by a conserved cytoplasmic catalytic kinase domain and a hydrophilic kinase sequence. The extracellular domain of VEGF receptor is constituted from seven immunoglobulin-like domains that are believed to be involved in the VEGF binding function.
The two most abundant and high affinity receptors of VEGF are VEGFR1 (Flt-1) and VEGFR2 (KDRIFlk-1). VEGFR1 is a gene isolated for the first time by Shibuya et al., and this is called as fms-like tyrosine kinase (Flt-1) due to its structural similarity (Non-Patent Document 1). On the other hand, VEGFR2 has a lower affinity to VEGF as compared to VEGFR1; however, autophosphorylation occurs at a high level and the kinase activity of VEGFR2 is approximately of the same degree as other representative receptor kinases (Non-Patent Document 2). A mouse homolog of KDR has an 85% amino acid homology with KDR, which is called as flk-1 (fetal liver kinase-1) (Non-Patent Document 3). VEGFR2 which is expressed in endothelial cells and which is directly involved in pathological angiogenesis has little involvement in the development of inflammatory diseases. On the other hand, VEGFR1 which is expressed in monocytes and macrophage system is suggested to be deeply involved in inflammation via the mobilization and activation of the function of inflammatory cells.
Patent Document 1 discloses that VEGF expression is inhibited by using a certain kind of anti-sense oligonucleotide which targets a VEGF RNA. Non-Patent Document 4 discloses that the binding of VEGF to its receptor is inhibited by using a particular VEGF specific high affinity RNA aptamer. Patent Document 2 discloses that a certain kind of anti-VEGF receptor monoclonal antibody is used to neutralize the effect of VEGF on the endothelial cells. However, any base sequence of a nucleic acid aptamer that specifically binds to a VEGF receptor has not been reported.
For the screening of a novel aptamer specifically adsorbing to a protein or a cell, in vitro selection method is used as a most effective means. In particular, Systematic Evolution of Ligands by Exponential enrichment (SELEX) method is roughly divided into two steps: the selection of target nucleic acid molecules and the amplification of selected aptamers (for example, Patent Document 3). Nucleic acid fragments with high affinity can be obtained by repeating the selection and amplification with an increased selection pressure. Moreover, in the recent years, various modifications have been made, and superior methods and the like in efficacy and selectivity which allows the collection of aptamers with fewer number of cycles are reported. These aptamers have various advantages that cannot be found in antibodies such as the following: synthesis can be performed chemically and in a short period of time, the modification of molecules can be performed economically, the action mechanism is simple, and hardly any immunogenicity is reported, which are additional advantages other than the high-affinity and specificity to a target that are features of an antibody conventionally used in diagnosis and treatment.